Lipid raft membrane compartmentalization and MAGUK-family molecular scaffolds function as key organizers of protein and membrane trafficking, cytoskeletal reorganization and signal transduction in neuronal and epithelial cell junctions. Here we consider a potential role for the Dlghl MAGUK family member in organizing the cytoskeleton, lipid rafts and signal transducers at the T cell:APC synapticjunction. During the past funding cycle we demonstrated a requirement for the LckSHS domain in mediating TCR/costimulation induced: synaptic raft clustering; processive and sustained signaling; reduced required duration of TCR engagement; Erk activation; and IL-2 production. We became interested in Dlghl as a potential LckSH3 effector because we identified it as a LckSH3 ligand and demonstrated LckSH3:Dlghl interactions as essential for Dlghl raft microdomain membrane localization. Moreover, we have recently identified WASp, Erk-1 and p38 as additional Dlghl ligands and suggest they may function as Dlghl(rLck) effectors. In this proposal, we design experiments aimed at assessing a potential role for Dlghl in TCR/costimulator induced signal transduction and elucidating the molecular basis of Dlghl activity. Furthermore, we consider the possibility that distinct T cell subsets differentially capitalize on Dlghl activities to generate synapses uniquely suited for effecting particular functions. To address these issues we capitalize on a three pronged approach involving siRNA mediated Dlghl knockdown, Dlghl over-expression/re-expression and dlghl gene knockout. We include analysisof Dlghl activity in the BI-141 T hybridoma, CD4+ 5CC7 and CD8+ OT-1 TCR transgenic T cells and in developing T cell subsets. We predict that direct comparison of Dlghl scaffolds withindistinct developing and T effector populations will elucidate novel TCR signal transduction mechanisms and molecular details involved in specializing synapses. Specifically, we propose the following: Arm 1) To investigate potential roles for Dlghl in antigen-induced T cell signal transduction, immune synapse assembly, and effector function; Aim 2) To determine the molecular basis of Dlghl activity in T cells; and Aim 3) To determine whether developing and effector T cells (differentially)rely on Dlghl scaffolding activities. Our studies will likely lead to a better understanding of how TCR signals are regulated to mediate functional outcome, which is essential to the design of therapeutics aimed at predictably modulating particular TCR responses. Indeed, elucidation of molecular mediators of synaptic organization and signal transduction events will provide novel targets for therapeutics aimed at inhibiting unwanted T cell activation responses including autoimmunity, graft rejection, and T cell transformation. Conversely, facilitators of individual activation events could be used in the design of tumor or other vaccines aimed at potentiating responses against suboptimally presented antigens.